branchless

Transcriptional regulation

Decapentaplegic controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. The requirement for Dpp is revealed by two manipulations: (1) the overexpression of Dpp using a heat-shock promoter and (2) use of mutations in the Dpp receptors thickveins and punt. The failure of tracheal cells to receive the DPP signal from adjacent dorsal and ventral cells results in the absence of dorsal and ventral migrations. Ectopic Dpp signaling can reprogram cells in the center of the placode to adopt a dorsoventral migration behavior. The effects observed in response to ectopic Dpp signaling are also observed upon the tracheal-specific expression of a constitutive active Dpp type I receptor (TKV[Q253D]). The alterations in migration behavior are similar for constitutively active receptor and for Dpp ectopic expression, indicating that the Dpp signal is received and transmitted in tracheal cells to control their migration behavior. Whereas, lack of Dpp signaling results in a failure of tracheal cells to migrate along the dorsoventral axis without significantly affecting anterior migrations, ubiquitous Dpp signaling suppresses anterior migrations without interfering with dorsoventral migration (Vincent, 1997).

Dpp signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the Branchless (Bnl) guidance molecule, the ligand of the Breathless (Btl) receptor. spalt (sal) is strongly expressed in dorsal trunk cells in stage 14 embryos and is necessary for the directed anterior migration of these cells. sal is expressed in the dorsal trunk in punt and tkv mutant embryos, indicating that Dpp does not regulate sal expression. However, embryos in which the Dpp signaling pathway has been activated in all tracheal cells at the placode stage fail to accumulate Sal. This lack of Sal expression correlates with the absence of the dorsal trunk upon ectopic Dpp signaling. In contrast to sal the gene knirps is activated in the developing tracheal system in all the branches (dorsal branch, ganglionic branch, and lateral trunk) that are thought to be under the control of Dpp. kni expression is lost in tkv mutants; (kni expression only persists in the visceral branches of tkv mutants). kni expression is turned on in all tracheal cells after constitutive Dpp signaling. The requirement for Dpp for the correct expression of the ligand branchless was revealed using tkv and punt mutants. The dorsal-most patches of bnl expression which prefigure the formation of the dorsal branches, are severely reduced in punt mutant embryos and absent in tkv mutants. Thus, Dpp plays a dual role during tracheal cell migration. It is required to control the dorsal expression of the Bnl ligand. In addition, the Dpp signal recruits groups of dorsal and ventral tracheal cells and programs them to migrate in dorsal and ventral directions (Vincent, 1997).

A central issue in developmental biology is how the deployment of generic signaling proteins produces diverse specific outcomes. Drosophila FGF is used, only in males, to recruit mesodermal cells expressing the FGF receptor to become part of the genital imaginal disc. Male-specific deployment of FGF signaling is controlled by the sex determination regulatory gene doublesex. The recruited mesodermal cells become epithelial and differentiate into parts of the internal genitalia. These results provide exceptions to two basic tenets of imaginal disc biology -- that imaginal disc cells are derived from the embryonic ectoderm and that they belong to either an anterior or posterior compartment. The recruited mesodermal cells migrate into the disc late in development and are neither anterior nor posterior (Ahmad, 2002).

The extensive sexual dimorphisms of the genitalia and analia suggest that the genital disc is relatively enriched in genes expressed downstream of dsx. To identify such genes, a random collection of enhancer traps was screened for sex-specific expression patterns in late third instar genital discs. Enhancer trap insertions in the bnl and btl genes were both isolated as enhancer traps expressed in male but not female genital discs. The sex specificity and the spatial patterns of expression of these enhancer traps accurately reflect the expression of the bnl and btl genes in the genital disc. Of the three primordia that comprise the genital disc, bnl and btl are both expressed in only one: the A9-derived developing 'male' primordium. bnl and btl are also expressed in adjacent domains: bnl is expressed at the base of two bilateral bowl-like infoldings of the disc epithelium, while btl is expressed in a group of loosely packed cells that fills these bowls and extends over the anterior and ventral surfaces of the disc (Ahmad, 2002).

The juxtaposition of btl- and bnl-expressing cells suggested that their proximity to one another might be the result of FGF-mediated cell-cell signaling. The locations of btl-expressing cells in male genital discs were determined at different stages of larval development. At early third instar (70-75 hr after egg laying), while a few btl-expressing cells are associated with the external surface of the disc, none are detected inside the disc. In mid-third instar (89-99 hr AEL), the btl-expressing cells are lying on the external surface of the disc, as well as adjacent to, and filling shallow invaginations in the disc epithelium. And by late third instar (110-120 hr AEL), these invaginations have become much deeper and are completely filled by btl-expressing cells. Thus, these btl-expressing cells are not originally a part of the disc epithelium but are recruited into invaginations in the epithelium during the third instar. Unlike the disc epithelium, the btl-expressing cells in the third instar disc do not express escargot (esg), a classical marker for ectoderm-derived imaginal cells, indicating that the btl-expressing cells have a different origin than do the other cells of the disc. The btl-expressing cells are, in fact, mesodermal in origin and derived from the adepithelial cells associated with the genital disc (Ahmad, 2002).

A priori, there are two possible explanations for the male-specific expression of FGF. One possibility is that bnl is an A9-specific gene, being expressed only in males where the A9-derived primordium grows significantly. The other possibility is that bnl is a target of the sex determination hierarchy, being either repressed by the female-specific Dsx protein (Dsx^F) in females and/or activated by the male-specific Dsx protein (Dsx^M) in males. To distinguish between these possibilities, feminized (Tra protein-expressing) clones of cells were generated in the A9-derived primordium of wild-type male genital discs and the effects of these clones on bnl expression were examined. Whenever feminized clones overlapped domains of bnl expression, the expression of bnl was repressed, indicating that it is cell-autonomous regulation by the sex determination hierarchy that is responsible for the male-specific expression of bnl in the genital disc (Ahmad, 2002).

When a feminized clone completely eliminated bnl expression from one side of a male disc, the lobe lacking bnl expression looked flattened. This was a consequence of btl-expressing cells not migrating into this lobe in the absence of Bnl protein, showing that bnl expression is not simply sufficient, but also necessary for the recruitment of btl-expressing cells. This observation suggests that btl, unlike bnl, is not a target of the sex determination hierarchy, and that the male-specific presence of btl-expressing cells in the genital disc is solely a consequence of Bnl recruiting the btl-expressing cells (Ahmad, 2002).

To examine how dsx regulates bnl expression, bnl expression was examined in wild-type genital discs and discs lacking dsx function. bnl is expressed in the A9-derived primordium of a wild-type male disc, where Dsx^M is present, but is not expressed in the A8-derived primordium of a wild-type female disc, where Dsx^F is expressed. However, in a disc in which neither Dsx protein is expressed, both the A8 and A9 primordia proliferate and bnl expression is seen in both primordia. That the A8 primordium grows in both wild-type and dsx mutant females but bnl is expressed in the A8 primordium only when the Dsx^F protein is absent, implies that bnl expression is repressed in the female genital disc by the presence of Dsx^F protein (Ahmad, 2002).

The ectopic expression of bnl in the A8-derived 'female' primordia of discs lacking dsx function offers an explanation for a puzzling observation: while wild-type males have only two paragonia (mesodermally derived components of the male disc), dsx mutant flies often have as many as four paragonia-like structures. The finding that the ectopic expression of bnl in flies mutant for dsx results in btl-expressing cells from the ventral surface of the disc being recruited into two ectopic invaginating pockets in the A8-derived female primordium of the disc, in addition to the original bowls in the A9-derived primordium, suggests that these ectopic pockets of btl-expressing cells give rise to the supernumerary paragonia when taken together with the observation that the extra paragonia in dsx mutants arise from the female primordium (Ahmad, 2002).

It is concluded that the sex-specific deployment bnl in the genital disc depends on the sex of the individual bnl-expressing cells. Given that bnl is regulated cell autonomously by Dsx^F, an obvious question is whether the Dsx^F protein directly represses bnl. In this regard, it is noted that 0.7 kb and 1.6 kb upstream of the putative bnl transcriptional start site, there are clusters of 5 and 4 sites respectively with at most a 1 bp mismatch to the 13 bp consensus Dsx binding site sequence. This is reminiscent of the 3 Dsx binding sites in a 76 bp stretch of an enhancer for the Yolk protein (Yp) genes, the only known direct targets of dsx (Ahmad, 2002).

The Drosophila sex determination hierarchy acts at multiple levels to control sexual differentiation. Some terminal differentiation genes like the Yp genes are direct transcriptional targets of the Dsx proteins and are continuously subject to their regulation. In other cases, the direct targets of dsx appear to be genes involved in initiating the differentiation of sex-specific tissues; genes expressed subsequently in these sex-specific tissues are governed by a tissue differentiation program, rather than being directly controlled by the sex hierarchy. It seems likely that the targets through which dsx initiates formation of such sex-specific tissues will be the genes where information from several developmental hierarchies are integrated to direct the differentiation of tissues (Ahmad, 2002).

These results suggest that bnl is one of the genes used by the sex determination hierarchy to direct the construction of sex-specific tissues. Bnl recruits btl-expressing cells into the male genital disc, and the recruited cells eventually form the paragonia and vas deferens (another mesodermally derived gonadal organ), tissues that are present only in males. Moreover, three genes expressed in the paragonia, the male-specific transcripts (msts) 316, 355a, and 355b, have been shown to be regulated in a tissue-specific rather than sex-specific manner: while transcription of these three male-specific RNAs begins in the late pupal period, their expression is governed by the sex hierarchy acting earlier, during the third larval instar -- the period when the expression of bnl recruits the paragonia-forming btl-expressing cells into the male genital disc. Thus, the sex-specific expression of the msts is achieved by dsx acting through bnl to generate the sex-specific tissue, the paragonia, in which the msts are subsequently expressed.

bnl also appears to be a gene where information from other regulatory hierarchies and the sex determination hierarchy are integrated in the male genital disc. The genetic hierarchies that control pattern formation and confer positional identity in the thoracic imaginal discs have previously been shown to function analogously in the genital disc. The fact that the bnl expression domain is limited to two specific subsets of the ectoderm-derived disc epithelia in males implies that bnl is also regulated by these pattern formation hierarchies. One area of future exploration will be examining how this coordinated regulation of bnl by dsx and the genes involved in pattern formation is brought about (Ahmad, 2002).

An intriguing aspect of these findings is the gradual transition of the btl-expressing cells, upon recruitment into the male genital disc, from twi-expressing mesodermal cells to epithelial cells with septate junctions. It is not clear if this transformation is also a consequence of FGF signaling, or if it is brought about by a different process. However, three separate observations suggest a role for bnl and btl in this mesoderm-epithelial transition: (1) FGF signaling mediates this process in mice -- during kidney development, FGF2 and leukemia inhibiting factor (LIF) secreted from the epithelial ureteric bud induce the conversion of the undifferentiated mesoderm-derived metanephric mesenchyme to the epithelial tubular structures of the nephron; (2) the converse process can also be mediated by FGF signaling -- FGFR1 regulates the morphogenetic movement and cell fate specification events during gastrulation in mice; it orchestrates the epithelial to mesenchymal transition during morphogenesis at the primitive streak and specifies the mesodermal cell fate of these mesenchymal cells, and (3) stumps, a gene acting downstream of the FGFR-encoding btl, has its expression elevated in the btl-expressing cells undergoing the transition into epithelial cells in the genital disc (Ahmad, 2002 and references therein).

Finally, it is noted that there are striking parallels between the roles of the FGF in sexual differentiation in the fly and FGF9 in sexual differentiation in mice. FGF9 is required for testicular embryogenesis in mice, and in its absence, XY mice undergo male-to-female sex reversal. FGF9 is expressed in the early embryonic gonads of male mice, not in the gonads of female mice, and not in the mesonephros of either sex, while bnl is expressed in the male genital disc, not in the female genital disc, and not in the btl-expressing mesodermal cells that are recruited into the male disc. The mesonephric cells migrate into only the male gonads, and the btl-expressing cells are recruited only into the male genital disc. Exogenous FGF9 induces mesonephric cell migration into female gonads, while ectopic expression of bnl is sufficient to recruit the btl-expressing cells into the female primordium of a dsx disc. The btl-expressing cells are mesodermal in origin, eventually undergo a transition into epithelial cells, and give rise to the vascular paragonia and vas deferens. The mesonephros, too, is derived from the mesoderm, and mesonephric cell migration into the testis contributes to the vascular endothelial, myoepithelial, and peritubular myoid cell populations. Given that there is considerable variation in the earlier aspects of sex determination across species, these findings suggest a possible conserved role for FGF signaling in later aspects of sexual differentiation (Ahmad, 2002 and references therein).

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. This approach was validated by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Strong evidence is presented showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo (Solano, 2003).

the expression of 14 genes was studied that are localized close to the genomic DNA fragments isolated in the library and tested previously for their Engrailed-specific binding ability. The results are shown for four genes (frizzled2, hibris, branchless, frazzled) that are representative of the different pathways where engrailed seems to be involved. frizzled 2 expression is activated in the presence of (VP16-En) and repressed in the presence of En. This suggests that engrailed might act as a repressor on fz2 expression. hibris is expressed along the wing margin and in the presumptive region of wing vein L3 and L4 in wild type. This expression is slightly activated in the presence of (VP16-En), but strongly repressed when En is overexpressed, suggesting that hbs expression is regulated by engrailed in vivo. branchless is essentially expressed in a dorsal/posterior territory surrounding the wing pouch in wild type. In the presence of (VP16-En), several additional patches of bnl expression are detected within the wing pouch, whereas no activation of bnl is observed after wild type En overexpression. As expected, because MS1096 drives Gal4 expression only in the wing pouch, endogenous bnl expression outside the wing pouch is not affected, showing the specificity of the experiment. Finally, frazzled is slightly expressed in wild-type wing disc. This expression is activated when (VP16-En) is overexpressed, and repressed upon En overexpression (Solano, 2003).

In Drosophila, trunk visceral mesoderm, a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. These findings have been extended by examining a greater number of visceral mesodermal genes, including hedgehog and branchless. Each visceral mesodermal parasegment appears to be divided in the A/P axis into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and con expression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts a feedback effect on trachea and ectodermal derivatives (Hosono, 2003).

Metameric RNA expression of bnl, which encodes a ligand for Breathless FGF receptor, is first observed as 12 patches at mid stage 11. bnl RNA expression becomes homogeneous and then diminished during stage 12. tin is a homeobox gene that is required for dorsal mesodermal development. At early stage 10, tin is expressed throughout the dorsal mesoderm from which VM is derived. Metameric Tin expression becomes evident by early stage 11. Tin expression decreases during stage 12. Expression of bap, another homeobox gene required for VM development, can be monitored by bap 4.5#230; (bap-lacZ). Staining for Tin and bap-lacZ or bnl RNA indicates that tin, bap and bnl are co-expressed in VM-PS3-12 during stage 11; in VM-PS2, only bnl is expressed. Stage 11-12 VM also stains for Tin and VM-hh-lacZ. Tin and VM-hh-lacZ expression partially overlaps. VM-hh expression in the anterior terminal region of VM-PSs indicates that each tin/bnl/bap trio expression domain straddles the VM-PS boundary (Hosono, 2003).

In summary, VM-PSs in thorax and abdomen, respectively, are subdivided into five or six regions with respect to differential expression of VM-metameric genes at stages 11-12. Detailed analysis of VM-hh, bnl, tin and bap expression in addition to con indicates that trunk visceral mesodermal genes are classified into three distinct groups -- tin/bnl/bap, VM-hh and con -- and each VM-PS is subdivided into five or six regions, which become apparent during mid stage 11 to stage 12 (Hosono, 2003).

VM is presently considered to develop in two steps under the control of ectodermal Hh and Wg signals. First, by stage 10 (when four mesodermal primordia have become specified), VM competent or bap expression regions are promoted by hh but repressed by wg, via a direct targetor, slp. The second surge of hh and wg activity at stages 10-11 is responsible for subdividing VM-PSs into two regions: con positive and negative. These results indicate that the expression of four other VM-metameric genes, hh, tin, bnl and bap, is also regulated by the second surge of hh and wg activity at stages 10-11 (Hosono, 2003).

In view of morphological changes in a VM competent region and consideration of these findings on VM gene regulation, the following model for VM-PS cell specification is proposed. At stage 10 to early stage 11, anterior terminal cells of VM-PSs are presumed to be situated near an ectodermal AP border, where they are capable of continuously receiving Wg and Hh signals, and Wg confers competence on these cells to express tin/bnl/bap. Wg and Hh are responsible for inducing VM-hh, and Hh, for con expression. In the anterior-most cells, con expression is reduced, which would be expected in view of repression by high Wg signal. The different thresholds of hh for con and VM-hh expression may explain why the con area expands more posteriorly compared with that of VM-hh. Posterior terminal VM cells, when formed, are situated far from Wg expressed on the ectodermal PS border. But as they migrate posteriorly and close to the posteriorly neighboring AP border by early stage 11, they become capable of receiving Wg and acquire competence to express tin/bnl/bap. Thus, the tin/bnl/bap domain would appear regulated by spatially and temporally distinct Wg signals. The two-step induction of tin/bnl/bap expression is supported by experiments using the wg^ts mutant, where, either posterior or anterior expression within one patch can be differentially turned off. Indeed, a stepwise activation of tin/bnl expression is seen in VM-PSs around stage 11. tin and bnl metameric expression became apparent almost simultaneously at mid-stage 11, and preliminary experiments have shown that neither tin nor bnl misexpression can induce the ectopic expression of any other metameric genes examined here. Thus, tin and bnl expression might be initiated in a mutually independent manner (Hosono, 2003).

Reiterative bnl expression in VM is likely to be a determinant of the particular mode of visceral branches (VB) migration of the trachea, an ectodermal organ. The tip of VB first comes in touch with the vicinity of the posterior end of the tin/bnl/bap expressing alpha region, where all the five metameric genes examined are expressed. Bnl misexpression with VM-specific-GAL4 drivers induces VB misrouting and bifurcation, but neither hh misexpression nor transient loss of Hh activity during stage 11 has any effect on VB budding. BNL misexpression brings about no significant change in expression of tin, while restriction of the tin/bnl/bap expression domain using either dTCF-DeltaN or wg^ts causes a shift in the first VB/VM contact point. Furthermore, under a wg mutant condition, no change is detected in VM-hh or in con expression. Thus, only the bnl expression appears to be closely correlated with VB budding, strongly suggesting that BNL serves as a chemoattractant for initial VB migration (Hosono, 2003).

Tramtrack regulates different morphogenetic events during Drosophila tracheal development

Tramtrack (Ttk) is a widely expressed transcription factor, the function of which has been analysed in different adult and embryonic tissues in Drosophila. So far, the described roles of Ttk have been mainly related to cell fate specification, cell proliferation and cell cycle regulation. Using the tracheal system of Drosophila as a morphogenetic model, a detailed analysis of Ttk function was undertaken. Ttk is autonomously and non-autonomously required during embryonic tracheal formation. Remarkably, besides a role in the specification of different tracheal cell identities, it was found that Ttk is directly involved and required for different cellular responses and morphogenetic events. In particular, Ttk appears to be a new positive regulator of tracheal cell intercalation. Analysis of this process in ttk mutants has unveiled cell shape changes as a key requirement for intercalation and has identified Ttk as a novel regulator of its progression. Moreover, Ttk was defined as the first identified regulator of intracellular lumen formation and; it is autonomously involved in the control of tracheal tube size by regulating septate junction activity and cuticle formation. In summary, the involvement of Ttk in different steps of tube morphogenesis identifies it as a key player in tracheal development (Araújo, 2007).

As with the transcription factors Trh and Vvl, which are involved in orchestrating early events of tracheal development, Ttk plays a role in orchestrating several late tracheal events. Ttk69 has been found to act mostly as a repressor. This study identified Ttk targets that appear to be negatively regulated (such as mummy (mmy), encodes a UDP-N-acetylglucosamine pyrophosphorylase enzyme required for the synthesis of the building blocks of chitin, and escargot (esg) whereas others appear to be positively regulated (such as polychaetoid (pyd) and branchless (bnl). In this latter case, Ttk might be converted into a positive regulator, as already described during photoreceptor development (Araújo, 2007).

This study identified multiple tracheal requirements for Ttk. Interestingly, most of them depend on Ttk regulating events downstream of cell fate specification, at the level of cellular responses. Additionally, a few other requirements depend on cell fate specification, as has been described for most other functions of Ttk in other developmental situations. For instance, Ttk regulates fusion cell specification by acting as a target and mediator of Notch, as occurs during sensory organ development and oogenesis. Such regulation of Ttk by N might be post-transcriptional, as occurs during sensory organ development. Remarkably, it was found that, although Ttk is sufficient to repress esg expression in fusion cells, it might not be the only esg- and fusion fate-repressor, because absence of Ttk does not increase the number of Esg-positive cells, as does downregulating N. Other N targets might be redundant with Ttk, and such redundancy could reinforce N-mediated repression of fusion fate in positions in which inductive signals (such as Bnl, Dpp and Wg) are very high, particularly near the branch tips (Araújo, 2007).

Cell rearrangements during development are common to most animals and ensure proper morphogenesis. During tracheal development, many branches grow and extend by cell intercalation. Several cellular and genetic aspects of tracheal intercalation have been well described. However, targets of Sal (which inhibits intercalation) are currently unknown (Araújo, 2007).

This study identified Ttk as a new and positive regulator of intercalation. Ttk is involved in cell junction modulation by transcriptionally regulating pyd, the only junctional protein shown, so far, to affect intercalation. In fact, modulation of AJs has been proposed to play a role during intercalation. However, Pyd cannot be the only Ttk effector of intercalation, because the pyd mutant phenotype is much weaker than that of ttk mutants. Accordingly, it was found that, in ttk mutants, cells in branches that usually intercalate remain paired and cuboidal, and appear unable to change shape and elongate. Although other explanations could account for the impaired intercalation detected in ttk mutants, it is proposed that inefficient cell shape changes represent the main cause, and might prevent the proper accomplishment of several events, such as the sliding of cells, formation of a first autocellular contact and zipping up, thereby blocking intercalation. Hence, it is proposed that cell shape changes, particularly cell elongation, are an obligate requisite for different steps of intercalation. Other targets of Ttk might presumably be regulators or components of the cytoskeleton involved in cell shape changes. It is relevant to point out here that Ttk has also been proposed to regulate morphogenetic changes required for dorsal appendage elongation (Araújo, 2007).

How does Ttk relate to the known genetic circuit (Sal-dependent) involved in intercalation? Being a transcription factor, Ttk initially appeared as an excellent candidate to participate in this genetic network by regulating sal and/or kni expression. However, both these genes to be normally expressed in ttk mutants, and several differences were detected in the intercalation phenotype of ttk loss versus sal upregulation. For instance, although both situations block intercalation, cells expressing sal, unlike those lacking ttk, are still able to undergo a certain change in shape, from cuboidal to elongated. Therefore, the results fit a model in which Ttk acts in a different and parallel pathway to Sal during intercalation. Consistent with this model, it was found that Ttk is not sufficient to promote intercalation on its own, because its overexpression cannot overcome the inhibition of intercalation imposed by Sal in the DT. Finally, genetic interactions also favour this model, because it was found that: (1) ttk overexpression did not rescue lack of intercalation produced by sal overexpression (even though it rescued the intercalation defects of ttk mutants), and (2) absence of sal (by means of the constitutive activation of the Dpp pathway) does not overcome the intercalation defects of ttk mutants. Therefore, it is proposed that Ttk promotes intercalation by endorsing changes in cell shape, but absence of Sal is still required to allow other aspects of intercalation to occur (Araújo, 2007).

Tube size regulation is essential for functionality. It was found that Ttk is involved in such regulation. Tube expansion and extension relies on a luminal chitin filament that assembles transiently in the tracheal tubes. The metabolic pathway that leads to chitin synthesis involves several enzymes, among which are Mmy and krotzkopf verkehrt (Kkv, a Chitin synthase). In addition, other proteins are known to participate in the proper assembly and/or modification of the chitin filament, such as Knk, Rtv, Verm and Serp. SJs are also required to regulate tube size and it was proposed that they exert this activity, at least partly, via the control of the apical secretion of chitin modifiers. The current results revealed that ttk acts as a key gene in tube size control, playing at least two roles: it regulates chitin filament synthesis and septate junction (SJ) activity (Araújo, 2007).

SJ regulation by Ttk appears functional rather than structural: mild defects were detected in the accumulation of only some SJ markers and there was a loss of the transepithelial diffusion barrier, whereas accumulation of other markers and SJ localisation remained apparently unaffected. It is speculated that Ttk transcriptionally controls one or several SJ components that contribute to maintain the paracellular barrier and to control a specialised apical secretory pathway. As a result, chitin binding proteins such as Verm or Serp are not properly secreted (Araújo, 2007).

It was also found that mmy is transcriptionally regulated by Ttk. mmy tracheal expression positively depends on a mid-embryonic peak of the insect hormone 20-hydroxyecdysone. Therefore, it is proposed that Ttk and ecdysone exert opposing effects on chitin synthesis. Excess of mmy mRNA results in the abnormal deposition of the chitin filament, as occurs in ttk mutants. Defects in chitin deposition might lead to the irregular organisation of taenidia and the faint larval cuticle observed in ttk mutants. Strikingly, Ttk is also required for normal chorion production, which represents another specialised secreted layer (Araújo, 2007).

ttk mutants are defective in the formation of terminal and fusion branches. These defects are due, in part, to non-autonomous, secondary and/or pleiotropic effects of ttk. For instance, ttk mutants exhibit a dorsal closure defect, which prevents the approach and fusion of contralateral dorsal branches. Additionally, terminal and fusion branches depend on correct cell type specification, which did not reliably occur in ttk mutants. For instance, DSRF (Blistered) was missing in some presumptive terminal cells of ttk mutants, impairing terminal branch formation. These tracheal cell identity specification defects might be related to non-autonomous requirements of ttk. For instance, DSRF is not properly expressed in ttk mutants because of an abnormal expression of its regulator, Bnl (Araújo, 2007).

It is important to note that, in spite of these non-autonomous and cell fate specification defects, two pieces of evidence indicate that ttk also plays a specific and autonomous role in the formation of terminal and fusion tubes. First, markers for fusion and terminal cell specification were expressed in many tracheal cells of ttk mutants, but yet most of these cells did not form terminal or fusion branches. Second, only the tracheal expression of ttk in ttk mutants (but not the constitutive activation of the btl pathway, which regulates the terminal and fusion identity) was able to restore the formation of terminal branches (Araújo, 2007).

A common feature of terminal and fusion branches is that they both display intracellular lumina that lack detectable junctions. The cellular events that precede the formation of fusion and terminal branches differ, but the mechanisms by which their intracellular lumina form has been proposed to be comparable. It was found that, in ttk mutants, terminal and fusion cells engage in the correct cellular changes before intracellular lumen formation. However, neither of these two cell types finalised the cellular events leading to tube formation. It has been proposed that the lumen of terminal and fusion branches forms by the coalescence of intracellular vesicles that use a 'finger' tip provided by the neighbouring stalk cell as a nucleation point. Interestingly, it was found that vesicles containing luminal material are less abundant in ttk mutants. These observations suggest a new role for Ttk in the formation of intracellular lumina in distinct cell types. Intracellular lumen formation also occurs in other branched tubular structures, such as in vertebrate endothelial cells and in the excretory cell of Caenorhabditis elegans, presumably by the coalescence of vesicles. Importantly, a crucial role for vesicle formation and their fusion during intracellular tube formation has been demonstrated (Araújo, 2007).

ttk is the first gene described to be involved in intracellular lumen formation during tracheal development. Possible targets of Ttk might be genes related to the apical surface and the underlying cytoskeleton, because several of these genes are involved in C. elegans excretory canal formation. Additionally, genes involved in intracellular vesicle trafficking might also be good candidates. In this respect, several abnormalities have been detected in ttk mutants that might reflect defects in vesicle trafficking (Araújo, 2007).

Targets of Activity

pointed and Serum response factor (also known as pruned, blistered and DSRF) are targeted by BNL. To define the role of bnl in later branching events, expression of secondary (pointed) and terminal (Srf) branch genes in bnl loss-of-function mutants was assayed. pnt and Srf fail to be expressed in the tracheal system of bnl mutants. In contrast, in embryos that ectopically express bnl, both markers are activated throughout the tracheal system; expressing cells later give rise to secondary and terminal branches. These results support the hypothesis that bnl expression near the ends of the primary branches not only guides primary branch outgrowth, but also activates the program of secondary and terminal branching in cells at these positions (Sutherland, 1996).

adrift is expressed in the leading cells of growing tracheal branches, near clusters of branchless FGF-expressing cells and in a pattern very similar to that of several known branchless-induced genes including pointed, DSRF/pruned and sprouty. This suggested that adrift expression might also be induced by the bnl signaling pathway. Expression of an adrift lacZ reporter was examined in embryos mutant for four components of the branchless pathway: bnl, breathless, pnt and pruned. Initial expression of the adrift reporter in stage 11 tracheal cells is normal in all four mutants, but subsequent expression in the leading cells of the branches is absent in bnl, btl and pnt mutants. Expression in pruned mutants is unaffected. In a complementary experiment in which bnl was misexpressed under the control of the hsp70 promoter, expression of the adrift reporter expands to include additional cells in each branch. Thus, the Branchless FGF pathway induces adrift expression in the leading cells of tracheal branches, and this induction requires the bnl FGF, the btl FGF receptor and the pointed ETS domain transcription factor (Englund, 1999).

blistered is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the blistered regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signaling pathway, is sufficient to induce blistered expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signaling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory (Nussbaumer, 2000).

A 500 bp enhancer element (TCE) has been isolated whose activity reproduces the blistered expression pattern in terminal tracheal cells, with respect to its temporal, spatial and regulatory cues. The TCE is the first enhancer identified in Drosophila that responds to FGF signaling. It has been reported that the FGF signaling cascade activates the MAP kinase pathway and that the Ets-domain containing protein Pnt is a target for the ERK-MAP kinase. Therefore, the expression of the TCE is likely to be controlled by FGF signaling which, through the ERK-MAP kinase pathway, activates a Pnt-DNA-bound complex in conjunction with other factors. Further dissection of the enhancer and the identification of the individual DNA sites and the relevant transcription factors should help to elucidate how FGF triggers specific nuclear responses. Interestingly, during early mesoderm induction in Xenopus laevis, an Ets-SRF complex has been implicated in transducing FGF signaling. Therefore, blistered might not only be a target gene whose transcription is activated in response to FGF signaling, but it might also encode a protein that assembles into a complex to integrate the FGF signal. However, the TCE does not require Blistered itself for signal induction since it is still active in a blistered loss-of-function mutant. Nevertheless, other putative target genes induced via FGF signaling in the terminal tracheal cells could require the activation of an Ets-Blistered-DNA complex (Nussbaumer, 2000).

Protein Interactions -

Breathless is the FGF receptor that serves to transduce branchless signals to the interior of the cell. breathless-branchless double mutants exhibit a tracheal phenotype similar to either one's loss-of-function mutation, as expected if the two function in the same signaling pathway. Reduction in the level of breathless can exacerbate the bnl-signaling defects, suggesting that bnl is insufficient in the haploid (single gene copy) state. A constitutively activated form of the BTL receptor can partially ameliorate the effect of the absence of bnl. Under such conditions there is a modest restoration of branching, reflecting the that the normal spatial distribution of activated receptor is not restored. BNL can activate the BTL receptor in vivo. In wild type extracts, a low level of phosphorylated BTL is detected, presumably due to its activation by endogenous BNL. In transgenic embryos that express bnl throughout the body, the level of phosphorylated BTL is increased about 8 fold (Sutherland, 1996).

The Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. sugarless and sulfateless mutant embryos are shown to have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. sfl and sgl mutants are shown to phenocopy the mesoderm migration defect associated with loss of heartless function and sfl and sgl are required for Btl-dependent tracheal cell migration. Moreover, both Heartless- and Breathless-dependent MAPK activation are significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. Overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization (Lin, 1999).

There are two well characterized HSPGs in Drosophila: Dally, a Glypican-like cell surface molecule that has been implicated in both Decapentaplegic and Wg signaling, and a transmembrane proteoglycan related to the vertebrate Syndecan family. It has been suggested that syndecans particpate in signaling by vertebrate FGFRs, although other HSPGs may also be involved in this process. It is also possible that different HSPGs could be specific for particular FGF ligand-receptor combinations in individual tissues or at distinct developmental stages. Genetic analysis in Drosophila should provide a useful approach for addressing these important questions (Lin, 1999 and references).

Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, this study characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. It was found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts (Kamimura, 2006; full text of article).

It was asked whether FGF signaling is impaired in mutant animals using an antibody that specifically recognizes the diphosphorylated form of MAP kinase (dpMAPK). In wild-type embryos, dpMAPK is detected in the tracheal placodes at stage 10, reflecting activation of Egfr. This dpMAPK signal was not diminished in the Hs2st; Hs6st embryos, showing that Egfr signaling is not affected by the double mutations. At stage 12, wild-type embryos show a strong dpMAPK signal in the migrating tip cells of each primary branch due to activation of FGF signaling. In contrast, the btl-dependent MAPK activation in the tip cells is disrupted in the Hs2st; Hs6st embryos. In situ RNA hybridization experiments revealed that bnl expression is not altered in the double mutant embryos, confirming that the branching defects observed in the double mutants are caused by disruption of FGF reception but not FGF expression. These results showed that HS with neither 2-O nor 6-O sulfate groups lost the ability to mediate Btl signaling (Kimimura, 2006).

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